(1) To develop animal, especially nonhuman primate, models that mimic human disease: We have established nonhuman primate (NHPs) models using Cynomolgus macaques for several influenza A viruses including the newly emerged H1N1 swine-origin influenza virus (SOIV), H1N1 and H3N2 seasonal influenza strains, H2N2 pandemic strain, and an H2N3 swine influenza strain. We used a recently established infection route that combined oral, intranasal, ocular and intratracheal inoculation of a total of 7x107 plaque forming units or tissue culture dose 50 units (TCID50). In general, the models differ in virus replication, clinical symptoms, disease progression and pathology. However, they do seem to mimic disease quite well as compared to human infections with the above mentioned strains. Seasonal influenza viruses H1N1 &H3N2 lead to either asymptomatic or fairly mild disease in Cynomolgus macaques. Shortly after infection animals show reduced food intake and occasionally mild respiratory signs. Gross pathological changes are limited to a few areas in the lungs. Histopathological investigations of those areas reveal alveolar edema and fibrin, hyaline membrane formation and type II alveolar pneumocyte hyperplasia. Animals recover quickly form the infection and clear virus within the first few days. There is limited evidence for virus shedding from mucosal membranes of the upper respiratory tract. collaborators: Drs. Taubenberger, DeLeo, Katze, Kawaoka, Kobinger, and Richt The pandemic H2N2 strain A/Singapore/1/57 (H2N2) leads to a moderate infection compared to the seasonal strains with moderate clinical symptoms and respiratory signs. Lung infiltrates, gross pathology and histopathology are in general slightly enhanced compared to the infections with seasonal viruses. Animals clear the infection with a delay and fully recover. There is limited evidence for virus shedding from mucosal membranes of the upper respiratory tract. collaborator: Dr. Richt We have analyzed a total of three different strains of the newly emerged SOIV. Interestingly, we observed different degrees of clinical severity, gross pathology, lung infiltration and histology. One of the Mexican strains isolated from a moderately sick human A/Mexico/4108/2009 (H1N1) behaved similar to the pandemic H2N2 strain. The California isolate from a sick boy with moderate disease A/California/4/2009 (H1N1) was more severe and the Mexico strain from a cluster of severe cases A/Mexico/4487/2009 (H1N1) was severe in clinical disease and pathology. All animals survived the infection but the animals with the severe infection had not completely cleared virus at the end of the study (day 14 post infection). This clearly indicates that a variety of SOIV strains co-circulate with different pathogenic potential. collaborators: Drs. Katze, Kawaoka, Kobinger, Richt, and Taubenberger Infection with the swine H2N3 virus A/swine/MS/06 (H2N3) was also more severe and was similar to the disease caused by the California strain of SOIV. Thus, this newly described swine virus has potential to infect primates and could potentially also infect humans. collaborator: Dr. J. Richt. Overall, we have established nonhuman primate (Cynomolgus macaque) models for seasonal and pandemic influenza A virus strains which nicely mimic influenza-associated disease in humans infected with the different influenza viruses. In general, influenza A viruses cause disease with different degrees of severity. The use of nonhuman primate disease models will in future support research on intervention strategies. (2) To identify and characterize determinants of pathogenicity in these animal models: To better understand the features that define highly lethal influenza viruses, we exploited a technology for the artificial generation and modification of influenza viruses (reverse genetics system). Using this technology, we have generated a consensus pandemic SOIV (H1N1) virus (parent virus). In order to study virulence of the different strains (isolates) and the mechanisms of pathogenesis we have generated selected recombinant viruses and characterized those mutants in vitro. For this we have compared the genome sequences of two early circulating SOIV strains with different pathogenicity in humans;A/Mexico/4108/2009 (M4108) (isolated from mild case) and A/Mexico/4487/2009 (M4487) (isolated from a cluster of severe cases). Sequence comparison revealed amino acid changes in 5 genomic segments, PB2, PA, HA, NP and M. The recombinant viruses were generated on the background of M4108 by replacing the corresponding genomic segment using the reverse genetics system. We are currently in the process to perform in vivo pathogenesis studies in ferrets. Subsequently, selected recombinants will be further tested in (Cynomolgus macaques). (3) To identify potential targets for intervention (antivirals and therapeutics): The inherent virulence property of the 1918 virus together with the current pandemic of the emerged SOIV (H1N1) influenza virus and the continued pandemic potential posed by circulation of the highly virulent H5N1 virus indicated the need for evaluating antiviral options against infections with virulent influenza viruses. Therefore, the recently developed Cynomolgus macaque model for the 1918 influenza virus was used for a drug efficacy study. We demonstrated that oseltamivir phosphate is effective in preventing severe disease in Cynomolgus macaques caused by the 1918 influenza virus if given prophylactically. Efficacy was reduced in a treatment regime through emergence of oseltamivir-resistant mutants that lead to death of one of four animals. This emphasizes the importance of implementing combination therapy and vaccination strategies early in a pandemic such as the current one caused by the emerged swine-origin H1N1 influenza virus. (4) To develop cross-protective vaccines and test their efficacy in the developed animal models. Our vaccine efforts have started in collaboration with Dr J Roses laboratory (New Haven, Connecticut, USA) using the rVSV vaccine vectors. The emergence in 1997 and continuance today of a highly lethal H5N1 avian influenza virus (AIV) causing human disease has raised concern about an impending pandemic and the need for a vaccine to prepare for such an occurrence. We have generated and characterized VSV-based vaccines that express the HK/156 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Post-challenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates. In the future, we are planning to use these vectors in the established nonhuman primate models. Expectations The studies will define determinants of pathogenicity by studying mutant influenza A viruses in vitro and in vivo (mouse, ferret, NHP models). We will further develop cross-protective vaccine platforms that could be applied in emergency response situations. Significance A better understanding of the underlying features of highly pathogenic influenza strains and the development of cross-protective vaccines are urgently needed to prepare for the emergence and control of highly lethal influenza viruses. Therefore, the proposed studies are directly associated with the primary mandates of NIAID, NIH, namely the response to emerging/re-emerging infectious diseases. The results are expected to have direct impact on national and international public health.